Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.     

cAMP regulates Ca2+-dependent exocytosis of lysosomes and lysosome-mediated cell invasion by trypanosomes.

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a beta-adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+]i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.     

Dynamics of endocytic traffic of Entamoeba histolytica revealed by confocal microscopy and flow cytometry

Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to obtain nutrients and, when induced to express invasive behavior, as a means of ingesting and processing host cells and tissue debris. E. histolytica trophozoites were grown in liquid axenic medium that contained fluorescently labeled fluid-phase markers, so that the kinetics of uptake, the transit of loaded endosomes through the cytoplasm, and the time of release of the markers could be monitored by flow cytometry. Confocal microscopy of live trophozoites revealed uptake of fluid by avid macropinocytosis and the occurrence of fusion between young and older endosomes, as well as between pinosomes and phagosomes containing bacteria. Endosomes were rapidly acidified, then gradually neutralized; finally, indigestible material was released. Transit of endosomes containing fluid-phase markers required about 2 h. Uptake and release of fluid-phase markers were impaired by drugs that inhibited actin dynamics and actin-myosin interaction; uptake was also impaired by inhibition of PI 3-kinase. A striking feature of the trophozoites was the great heterogeneity of their endocytic behavior.     

Rab11B small GTPase regulates secretion of cysteine proteases in the enteric protozoan parasite Entamoeba histolytica

Vesicular trafficking plays a pivotal role in the virulence of the enteric protozoan parasite Entamoeba histolytica. In the present study, we showed that one isotype of the small GTPase Rab11, EhRab11B, plays a central role in the secretion of a major virulence factor, cysteine proteases. EhRab11B did not colocalize with markers for the endoplasmic reticulum, early endosomes and lysosomes, but was partially associated with non-acidified vesicles in the endocytic pathway, likely recycling endosomes. Overexpression of EhRab11B resulted in a remarkable increase in both intracellular and secreted cysteine protease activity, concomitant with an augmentation of cytolytic activity as demonstrated by an increased ability to destroy mammalian cells. The oversecretion of cysteine proteases with EhRab11B overexpression was neither sensitive to brefeldin A nor specific to a certain cysteine protease species (e.g. CP1, 2 or 5), suggesting that these three major cysteine proteases are trafficked via an EhRab11B-associated secretory pathway, which is distinct from the classical brefeldin-sensitive pathway. Overexpression of EhRab11B also enhanced exocytosis of the incorporated fluid-phase marker, supporting the notion that it is involved in recycling. This is the first report demonstrating that Rab11 plays a central role in the transport and secretion of pathogenic factors.     

Dendritic cell-mediated HIV-1 transmission to T cells of LAD-1 patients is impaired due to the defect in LFA-1

Background

Murine Leukemia Virus (MLV) assembly has been long thought to occur exclusively at the plasma membrane. Current models of retroviral particle assembly describe the recruitment of the host vacuolar protein sorting machinery to the cell surface to induce the budding of new particles. Previous fluorescence microscopy study reported the vesicular traffic of the MLV components (Gag, Env and RNA). Here, electron microscopy (EM) associated with immunolabeling approaches were used to go deeply into the assembly of the "prototypic" MLV in chronically infected NIH3T3 cells.

Results

Beside the virus budding events seen at the cell surface of infected cells, we observed that intracellular budding events could also occur inside the intracellular vacuoles in which many VLPs accumulated. EM in situ hybridization and immunolabeling analyses confirmed that these latter were MLV particles. Similar intracellular particles were detected in cells expressing MLV Gag alone. Compartments containing the MLV particles were identified as late endosomes using Lamp1 endosomal/lysosomal marker and BSA-gold pulse-chase experiments. In addition, infectious activity was detected in lysates of infected cells.

Conclusion

Altogether, our results showed that assembly of MLV could occur in part in intracellular compartments of infected murine cells and participate in the production of infectious viruses. These observations suggested that MLV budding could present similarities with the particular intracellular budding of HIV in infected macrophages.     

Coxiella burnetii survival in THP-1 monocytes involves the impairment of phagosome maturation: IFN-gamma mediates its restoration and bacterial killing

The subversion of microbicidal functions of macrophages by intracellular pathogens is critical for their survival and pathogenicity. The replication of Coxiella burnetii, the agent of Q fever, in acidic phagolysosomes of nonphagocytic cells has been considered as a paradigm of intracellular life of bacteria. We show in this study that C. burnetii survival in THP-1 monocytes was not related to phagosomal pH because bacterial vacuoles were acidic independently of C. burnetii virulence. In contrast, virulent C. burnetii escapes killing in resting THP-1 cells by preventing phagosome maturation. Indeed, C. burnetii vacuoles did not fuse with lysosomes because they were devoid of cathepsin D, and did not accumulate lysosomal trackers; the acquisition of markers of late endosomes and late endosomes-early lysosomes was conserved. In contrast, avirulent variants of C. burnetii were eliminated by monocytes and their vacuoles accumulated late endosomal and lysosomal markers. The fate of virulent C. burnetii in THP-1 monocytes depends on cell activation. Monocyte activation by IFN-gamma restored C. burnetii killing and phagosome maturation as assessed by colocalization of C. burnetii with active cathepsin D. In addition, when IFN-gamma was added before cell infection, it was able to stimulate C. burnetii killing but it also induced vacuolar alkalinization. These findings suggest that IFN-gamma mediates C. burnetii killing via two distinct mechanisms, phagosome maturation, and phagosome alkalinization. Thus, the tuning of vacuole biogenesis is likely a key part of C. burnetii survival and the pathophysiology of Q fever.     

Toxin pores endocytosed during plasma membrane repair traffic into the lumen of MVBs for degradation

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.     

Interaction of microbial pathogens with host exocytic pathways

Many microbial pathogens co‐opt or perturb host membrane trafficking pathways. This review covers recent examples in which microbes interact with host exocytosis, the fusion of intracellular vesicles with the plasma membrane. The bacterial pathogens Listeria monocytogenes and Staphylococcus aureus subvert recycling endosomal pathways of exocytosis in order to induce their entry into human cells. By contrast, entry of the protozoan pathogen Trypanosoma cruzi or the virus adenovirus into host cells involves exploitation of lysosomal exocytosis. Toxins produced by Bacillus anthracis or Vibrio cholerae interfere with exocytosis pathways mediated by the GTPase Rab11 and the exocyst complex. By doing so, anthrax or cholera toxins impair recycling of cadherins to cell–cell junctions and disrupt the barrier properties of endothelial cells or intestinal epithelial cells, respectively. Uropathogenic Escherichia coli (UPEC) is expelled from bladder epithelial cells through two different exocytic routes that involve sensing of bacteria in vacuoles by host Toll‐like receptor 4 (TLR4) or monitoring of the pH of lysosomes harbouring UPEC. The TLR4 pathway is mediated by multiple Rab GTPases and the exocyst, whereas the other pathway involves exocytosis of lysosomes. Expulsion of UPEC through these pathways is thought to benefit the host.     

Lysosomal exocytosis: An important event during invasion of lamp deficient cells by extracellular amastigotes of Trypanosoma cruzi

Trypanosoma cruzi is an obligate intracellular organism in vertebrate hosts. Lysosomes are involved in parasite invasion. LAMP-1 and LAMP-2 are the most abundant glycoproteins of the lysosomal membrane. This study is the first report on the invasion of T. cruzi extracellular amastigotes (EA) in single LAMP-1 or LAMP-2 knockouts, respectively, or in two independent LAMP-1/2 double-knockout cell lines. When compared to their respective wild type clones, the EA show higher infectivity in LAMP-2 knockouts, but no difference was seen in LAMP-1 knockout cells. Similarly, EA invasion rate was higher for one of the double knockout clones but not for the other. Higher lysosomal exocytosis correlated with a higher invasion rate and early lysosomal marker acquisition. These findings suggest that lysosomal exocytosis is important to EA cell invasion. Also, phagolysosome maturation in knockout cell lines differed from previous results revealing that EA enter cells by a mechanism other than receptor-mediated phagocytosis.     

Host Cell Autophagy Modulates Early Stages of Adenovirus Infections in Airway Epithelial Cells

Human adenoviruses typically cause mild infections in the upper or lower respiratory tract, gastrointestinal tract, or ocular epithelium. However, adenoviruses may be life-threatening in patients with impaired immunity and some serotypes cause epidemic outbreaks. Attachment to host cell receptors activates cell signaling and virus uptake by endocytosis. At present, it is unclear how vital cellular homeostatic mechanisms affect these early steps in the adenovirus life cycle. Autophagy is a lysosomal degradation pathway for recycling intracellular components that is upregulated during periods of cell stress. Autophagic cargo is sequestered in double-membrane structures called autophagosomes that fuse with endosomes to form amphisomes which then deliver their content to lysosomes. Autophagy is an important adaptive response in airway epithelial cells targeted by many common adenovirus serotypes. Using two established tissue culture models, we demonstrate here that adaptive autophagy enhances expression of the early region 1 adenovirus protein, induction of mitogen-activated protein kinase signaling, and production of new viral progeny in airway epithelial cells infected with adenovirus type 2. We have also discovered that adenovirus infections are tightly regulated by endosome maturation, a process characterized by abrupt exchange of Rab5 and Rab7 GTPases, associated with early and late endosomes, respectively. Moreover, endosome maturation appears to control a pool of early endosomes capable of fusing with autophagosomes which enhance adenovirus infection. Many viruses have evolved mechanisms to induce autophagy in order to aid their own replication. Our studies reveal a novel role for host cell autophagy that could have a significant impact on the outcome of respiratory infections.     

Two motifs target Batten disease protein CLN3 to lysosomes in transfected nonneuronal and neuronal cells

Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs.     

Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event

Bacterial cell wall constituents are released from mycobacterial phagosomes and actively traffic within infected macrophages. Colocalization of fluorescently tagged bacterial moieties with endocytic tracers revealed the dynamic movement of released mycobacterial constituents into the endocytic network with accumulation in tubular lysosomal-like compartments. The released bacterial constituents not only penetrated the infected host cell but were also present in an extracellular microvesicular fraction. To identify the intracellular source of these exocytic compartments, released vesicular material was isolated from culture supernatants by differential ultracentrifugation and characterized by Western blot and electron microscopy analyses. The presence of lysosomal membrane proteins and lysosomal proteases suggested that labeled mycobacterial cell wall constituents access a constitutive lysosomal exocytic pathway. An abundance of multilamellar extracellular compartments morphologically reminiscent of MHC class II-enriched compartments (MIIC) implicated a MHC class II transport pathway in the extracellular release of bacterial constituents. Increases in intracellular free calcium have previously been shown to trigger lysosomal exocytosis by inducing fusion of lysosomes with the plasma membrane. To test if an increase in calcium would stimulate exocytosis with release of mycobacterial constituents, infected macrophages were exposed to the calcium ionophore A23187. The ionophore triggered the release of a microvesicular fraction containing labeled bacterial moieties, implicating calcium-regulated lysosomal exocytosis as a trafficking pathway by which mycobacterial products are released from infected macrophages.     

Pathogen trafficking pathways and host phosphoinositide metabolism

Phosphoinositide (PI) glycerolipids are key regulators of eukaryotic signal transduction, cytoskeleton architecture and membrane dynamics. The host cell PI metabolism is targeted by intracellular bacterial pathogens, which evolved intricate strategies to modulate uptake processes and vesicle trafficking pathways. Upon entering eukaryotic host cells, pathogenic bacteria replicate in distinct vacuoles or in the host cytoplasm. Vacuolar pathogens manipulate PI levels to mimic or modify membranes of subcellular compartments and thereby establish their replicative niche. Legionella pneumophila , Brucella abortus , Mycobacterium tuberculosis and Salmonella enterica translocate effector proteins into the host cell, some of which anchor to the vacuolar membrane via PIs or enzymatically turnover PIs. Cytoplasmic pathogens target PI metabolism at the plasma membrane, thus modulating their uptake and antiapoptotic signalling pathways. Employing this strategy, Shigella flexneri directly injects a PI-modifying effector protein, while Listeria monocytogenes exploits PI metabolism indirectly by binding to transmembrane receptors. Thus, regardless of the intracellular lifestyle of the pathogen, PI metabolism is critically involved in the interactions with host cells.     

Mycobacterium and the coat of many lipids

Pathogenic Mycobacterium reside inside vacuoles in their host macrophages. These vacuoles fail to fuse with lysosomes yet interact with early endosomes. Glycoconjugates released by the intracellular bacilli traffic through the host cell and are released through exocytosis. These molecules represent both antigens for immune recognition and modulators of immune function. The molecules play key roles in the induction and maintenance of the granuloma, a tissue response that limits bacterial spread yet ensures persistence of the infection.     

Endosomes and Microtubles are Required for Productive Infection in Aquareovirus

Grass carp reovirus(GCRV), the genus Aquareovirus in family Reoviridae, is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection, the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches. We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH_4Cl, chloroquine and bafilomycin A1(Bafi). Confocal images indicated that GCRV particles could colocalize with Rab5, Rab7 and lysosomes in host cells. Further ultrastructural examination validated that viral particle was found in late endosomes. Moreover, disruption of microtubules with nocodazole clearly blocked GCRV entry, while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament. Notably, viral particles were observed to transport along microtubules by using QD-labeled GCRV. Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.     

Functional analysis of host factors that mediate the intracellular lifestyle of Cryptococcus neoformans

Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.     

Mechanism of Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction

Abstract: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.     

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella‐containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane‐bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood. Here, we identify Nischarin as a novel dual effector that can interact with members of Rac and Rab GTPase (Rab4, Rab14 and Rab9) families at different endosomal compartments. Nischarin interacts with GTP‐bound Rab14 and PI(3)P to direct the maturation of early endosomes to Rab9/CD63‐containing late endosomes. Nischarin is recruited to the SCV in a Rab14‐dependent manner and enhances acidification of the SCV. Depletion of Nischarin or the Nischarin binding partners—Rac1, Rab14 and Rab9 GTPases—reduced the intracellular growth of Salmonella. Thus, interaction of Nischarin with GTPases may regulate maturation and subsequent acidification of vacuoles produced after phagocytosis of pathogens.     

Effect of monensin on the neuronal ultrastructure and endocytic pathway of macromolecules in cultured brain neurons

1. The endocytic pathway of horseradish peroxidase (HRP) was investigated in the perikarya of cultured neurons by electron microscopy and enzyme cytochemistry. The tracer was observed in endocytic pits and vesicles, endosomes, multivesicular bodies, and lysosomes. It took approximate 15 min for the transfer of HRP from the exterior of the cell to the lysosomes. 2. Monensin induced distension of the Golgi apparatus and formation of intracellular vacuoles. When neurons were incubated with both monensin and HRP for 30 to 120 min, the number of HRP-labeled endosomes was greater than that in the monensin-free group, whereas the reverse was seen for HRP-positive lysosomes. The formation of HRP-positive lysosomes in monensin-treated cells was blocked by 47 to 79%. 3. These results indicate that the intracellular transport of the endocytosed macromolecule is pH dependent. It is also possible that the export of lysosomal enzymes is inhibited by monensin, resulting in an accumulation of the endosomes and a reduction of the lysosomes.